Therapeutic Restoration of Endothelial Glycocalyx in Sepsis.

Endothelial glycocalyx (EG) is disintegrated during sepsis. We have previously shown that this occurs very early in the course of sepsis and its prevention improves the survival of mice with sepsis. Here, we sought to investigate the possibility of pharmacologically accelerating the restoration of disintegrated EG in sepsis. We used a soilage injection model to induce polymicrobial sepsis in C57/BL6 mice and measured total body EG. En face aortic preparations were used for staining of markers of EG and atomic force microscopy was used to measure EG in vitro. In vitro studies were conducted in cultured endothelial cells either exposed to a lipopolysaccharide or enzymatically denuded of EG. Sulodexide (SDX), a heparin sulfate-like compound resistant to degradation by heparanase, accelerated EG regeneration in vitro and in vivo. The total volume of EG was drastically reduced in septic mice. Administration of SDX produced a dramatic acceleration of EG restoration. This effect, unrelated to any SDX-induced differences in microbial burden, was associated with better control of vascular permeability. Notably, SDX demonstrated not only a remarkable capacity for EG regeneration in vitro and in vivo but was also associated with improved animal survival, even when instituted 2 hours after induction of severe sepsis. In conclusion, 1) EG is disintegrated in sepsis, the event which contributes to high animal mortality; 2) pharmacologic acceleration of EG restoration can be achieved using SDX; and 3) SDX reduces vascular permeability, which is elevated in septic mice, and improves animal survival.

Quantitation of cell-associated borrelial DNA in the blood of Lyme disease patients with erythema migrans.

Bloodstream invasion is an important event in the pathogenesis of the more serious manifestations of Lyme disease. The number of spirochetes in the blood of infected patients, however, has not been determined, and, therefore, it is unknown whether the number of spirochetes can be correlated with particular clinical or laboratory features. This study was designed to measure the level of Borrelia burgdorferi in the plasma of Lyme disease patients and correlate these levels with selected clinical and laboratory findings. Nested and quantitative polymerase chain reaction (qPCR) was employed to detect cell-associated flaB gene DNA in the plasma of untreated early Lyme disease patients with erythema migrans (EM). Twenty-nine (45.3%) of 64 patients had evidence of B. burgdorferi in their plasma by at least one of the PCR methods. For the 22 qPCR-positive patients, the mean number of flaB gene copies per mL of plasma was 4,660, with a range of 414 to 56,000. The number of flaB gene copies did not significantly correlate with any of the clinical, demographic, or laboratory variables assessed. For reasons discussed, we suggest caution in extrapolating an estimate of the number of viable Borrelia in plasma from the observed number of flaB copies.

In vivo and in vitro studies on Anaplasma phagocytophilum infection of the myeloid cells of a patient with chronic myelogenous leukaemia and human granulocytic ehrlichiosis.

AIMS: The occurrence of human granulocytic ehrlichiosis (HGE) in a patient with chronic myelogenous leukaemia (CML) provided an opportunity to study whether Anaplasma phagocytophilum, the aetiological agent of HGE, infects mature or immature cells, both in vivo and in vitro. METHODS: Diagnosis of HGE was confirmed by culture, polymerase chain reaction (PCR), detection of intragranulocytic inclusions, and serology. The infection rates of different myelogenous stages of granulocytic differentiation were determined by microscopy. Anaplasma phagocytophilum infection of the bone marrow was analysed by PCR, culture, and microscopy. In addition, the in vitro growth of A phagocytophilum in the patient's granulocytes and in HL-60 cells (a promyelocytic leukaemia cell line) was compared. RESULTS: Pretreatment blood smears showed that mature granulocytic cells had a higher infection rate with A phagocytophilum than did immature cells. In the original inoculation of the patient's cells into HL-60 cells to isolate A phagocytophilum, the bacterium grew faster in the patient's leukaemic cells than in HL-60 cells. Anaplasma phagocytophilum inclusions were rarely seen in bone marrow granulocytes and PCR was negative. In vitro, two A phagocytophilum isolates grew faster in the patient's granulocytes than in HL-60 cells. CONCLUSIONS: The superior growth in CML cells compared with HL-60 cells suggests that A phagocytophilum preferentially infects mature granulocytes. The higher infection rate of the patient's mature versus immature granulocytes before treatment and the minimal level of infection of the patient's bone marrow support this. It is possible that the primary site of infection in HGE is the peripheral mature granulocytic population.

Laboratory diagnostic techniques for patients with early Lyme disease associated with erythema migrans: a comparison of different techniques.

Recently, a number of refinements in diagnostic modalities for detection of Borrelia burgdorferi infection have been developed. These include large-volume blood cultures, quantitative polymerase chain reaction (PCR) techniques, and 2-stage serologic testing. In the present study, we compared 6 diagnostic modalities in 47 adult patients who had a clinical diagnosis of erythema migrans. Quantitative PCR on skin biopsy-derived material was the most sensitive diagnostic method (80.9%), followed by 2-stage serologic testing of convalescent-phase samples (66.0%), conventional nested PCR (63.8%), skin culture (51.1%), blood culture (44.7%), and serologic testing of acute-phase samples (40.4%). Results of all assays were negative for 3 patients (6.4%). We conclude that the clinical diagnosis of erythema migrans is highly accurate in an area where B. burgdorferi is endemic if it is made by experienced health care personnel, but some patients with this diagnosis may not have B. burgdorferi infection. No single diagnostic modality is suitable for detection of B. burgdorferi in every patient with erythema migrans.

Characterization of Borrelia burgdorferi isolated from erythema migrans lesions: interrelationship of three molecular typing methods.

Genetic diversity among Borrelia burgdorferi isolates recovered from the skin of Lyme disease patients was assessed by ribosomal DNA (rDNA) spacer restriction fragment length polymorphism analysis, genomic restriction site polymorphism analysis, and plasmid content analysis. There was a significant association between the three rDNA spacer types, the six pulsed-field gel types, and plasmid content (P < 0.001). The association between distinct chromosomal and plasmid markers implies a clonal origin for each genotype.

Borrelia lonestari infection after a bite by an Amblyomma americanum tick.

Erythematous rashes that are suggestive of early Lyme disease have been associated with the bite of Amblyomma americanum ticks, particularly in the southern United States. However, Borrelia burgdorferi, the causative agent of Lyme disease, has not been cultured from skin biopsy specimens from these patients, and diagnostic serum antibodies usually have not been found. Borrelia lonestari sp nov, an uncultured spirochete, has been detected in A. americanum ticks by DNA amplification techniques, but its role in human illness is unknown. We observed erythema migrans in a patient with an attached A. americanum tick. DNA amplification of the flagellin gene flaB produced B. lonestari sequences from the skin of the patient that were identical to those found in the attached tick. B. lonestari is a probable cause of erythema migrans in humans.

Association of specific subtypes of Borrelia burgdorferi with hematogenous dissemination in early Lyme disease.

To investigate whether genetic diversity of Borrelia burgdorferi sensu stricto may affect the occurrence of hematogenous dissemination, 104 untreated adults with erythema migrans from a Lyme disease diagnostic center in Westchester County, New York, were studied. Cultured skin isolates were classified into 3 groups by a polymerase chain reaction amplification and restriction fragment length polymorphism (RFLP) method. A highly significant association between infecting RFLP type in skin and the presence of spirochetemia was found (P<.001). The same association existed for the presence of multiple erythema migrans lesions (P=.045), providing clinical corroboration that hematogenous dissemination is related to the genetic subtype of B. burgdorferi sensu stricto. There were no significant associations between RFLP type and seropositivity or clinical symptoms and signs except for a history of fever and chills (P=.033). These results suggest that specific genetic subtypes of B. burgdorferi sensu stricto influence disease pathogenesis. Infection with different subtypes of B. burgdorferi sensu stricto may help to explain differences in the clinical presentation of patients with Lyme disease.

Genetic diversity of Borrelia burgdorferi in lyme disease patients as determined by culture versus direct PCR with clinical specimens.

Two hundred seventeen isolates of Borrelia burgdorferi originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA intergenic spacer. Three major RFLP types were observed. Of the cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%) were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP types were obtained in 6.0% (13 of 217) of the cultures. Comparison of typing of B. burgdorferi performed directly on 51 patient skin specimens with typing of cultures originally isolated from the same tissue revealed that a much larger proportion of direct tissue samples had mixtures of RFLP types (43.1% by direct typing versus 5.9% by culture [P < 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of B. burgdorferi patient isolates as determined by cultivation differs from that assessed by PCR performed directly on patient tissue.

Lyme disease and ehrlichiosis.

This article provides the podiatric practitioner with current information about Lyme disease and ehrlichlosis. The material presented assists podiatrists, particularly those practicing in rural areas, in recognizing these tick-bone diseases and in making informed decisions about treatment. The steady increase of these "new and emerging infectious diseases" places additional importance on health care providers, such as podiatrists, to inform the public and help prevent the incidences of tick borne diseases.

Molecular typing of Borrelia burgdorferi from Lyme disease patients by PCR-restriction fragment length polymorphism analysis.

Ninety-three Borrelia burgdorferi isolates obtained from erythema migrans lesions or blood of Lyme disease patients in Westchester County, N.Y., between 1991 and 1994 were characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S-23S rRNA gene spacer. All isolates could be classified into three distinct RFLP types. Among the 82 skin biopsy isolates studied, 21 (25.6%) were type 1, 37 (45.1%) were type 2, and 21 (25.6%) were type 3. Three (3.7%) cultures contained a mixture of two isolates with distinct RFLP types. The 11 isolates cultured from blood showed a similar predominance of RFLP type 2 (6 of 11; 54.5%) relative to types 1 (2 of 11; 18.2%) and 3 (3 of 11; 27.3%). For one patient both skin and blood isolates were cultured, and RFLP analysis revealed that these isolates differed from one another. This study demonstrates that there is genotypic heterogeneity in B. burgdorferi strains infecting Lyme disease patients, and this typing approach may allow differentiation of isolates with various degrees of pathogenic potential.

Identification of novel insertion elements, restriction fragment length polymorphism patterns, and discontinuous 23S rRNA in Lyme disease spirochetes: phylogenetic analyses of rRNA genes and their intergenic spacers in Borrelia japonica sp. nov. and genomic group 21038 (Borrelia andersonii sp. nov.) isolates.

Borrelia spp. associated with Lyme disease possess an rRNA gene organization consisting of a single 16S rRNA gene followed by a spacer of several kilobases and a tandem repeat of a 23S (rrl)-5S (rrf) rRNA gene cluster. The restriction fragment length polymorphism (RFLP) patterns for these genes have been widely used to classify Lyme disease spirochete isolates. We analyzed the rRNA gene organization and sequences for two Ixodes ovatus isolates from Japan (IKA2 and HO14) and two group 21038 isolates associated with Ixodes dentatus ticks or rabbits from North America (isolates 21038 and 19857). This analysis revealed unique polymorphisms not previously described in other Lyme disease spirochete isolates. The molecular basis of these polymorphisms was determined by Southern blotting and PCR analyses. Only one continuous copy of the rrl-rrf gene cluster was identified in isolates IKA2, 19857, and 21038. The second rrl-rrf gene cluster is entirely absent from the IKA2 genome. In isolates 19857 and 21038, an intervening sequence is present, resulting in a fragment rrlB gene. The insertion site of this intervening sequence element differed in each isolate. While isolates 19857 and 21038 were found to carry a fragmented rrlB gene, they lacked rrfB. To determine if these rRNA polymorphisms were indicative of an underlying phylogenetic divergence, sequence analysis of the 16S rRNA (rrs) genes was conducted. The phylogenies inferred from rrs sequence analysis suggest that the polymorphisms resulted from recent mutational events. In addition, the phylogenetic analyses also support the proposed species status of Borrelia japonica sp. nov. and indicate that isolates of genomic group 21038 belong to a previously undescribed species for which we propose the nomenclature Borrelia andersonii sp. nov.

rRNA operon multiplicity in Escherichia coli and the physiological implications of rrn inactivation.

Here we present evidence that only five of the seven rRNA operons present in Escherichia coli are necessary to support near-optimal growth on complex media. Seven rrn operons are necessary, however, for rapid adaptation to nutrient and temperature changes, suggesting it is the ability to adapt quickly to changing environmental conditions that has provided the selective pressure for the persistence of seven rrn operons in E. coli. We have also found that one consequence of rrn operon inactivation is a miscoordination of the concentrations of initiation factor IF3 and ribosomes.

Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis.

The etiologic agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, has been isolated from many biologic sources in North America and Eurasia, and isolates have been divided into three distinct genospecies (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii). In order to explore the possible association of genospecies with disease manifestation, 60 isolates of B. burgdorferi sensu lato were subjected to 5S rDNA-linked restriction fragment length polymorphism (RFLP) analysis. The results confirmed earlier studies which indicated that virtually all North American isolates are B. burgdorferi sensu stricto, whereas Eurasian strains fall into all three genospecies. Thirty-five isolates were further characterized by PCR amplification of a region of the 16S-23S rDNA spacer and HinfI digestion of the products. This method resulted in the subdivision of B. burgdorferi sensu stricto into two distinct PCR-RFLP types. In contrast, B. garinii isolates all displayed an identical pattern. Additionally, a number of previously unclassified North American isolates (25015, DN127, 19857, 24330) showed distinctively different PCR-RFLP patterns. The application of this method for the typing of uncultured B. burgdorferi directly in biologic samples was demonstrated by analysis of several field-collected Ixodes scapularis tick specimens. The described PCR-RFLP technique should allow for the direct and rapid molecular typing of B. burgdorferi-containing samples and facilitate studies of the relationship between spirochete genotype and clinical disease.

Sequence analysis of the ribosomal RNA operon of the Lyme disease spirochete, Borrelia burgdorferi.

An 11,955-bp region of the Borrelia burgdorferi chromosome containing all the genes encoding ribosomal RNA (rRNA) has been sequenced. The region contains a single gene encoding 16S rRNA and two genes encoding the 23S and 5S rRNAs. The sizes of the 16S, 23S and 5S rRNAs encoded by these genes are 1537, 2926 and 112 nucleotides, respectively. In addition, the genes encoding tRNA(Ala) and tRNA(Ile) are located in the intergenic spacer between the 16S and 23S rDNAs. The tDNAs do not encode the common CCA 3' end which presumably must be added posttranscriptionally. All the genes are present in the same orientation, except for that encoding tRNA(Ile), which is transcribed from the opposite strand. The latter implies that the rDNAs are not transcribed as a single unit. The location of putative promoters and termination signals in the sequence suggest that the 16S rRNA and tRNA(Ala) are transcribed as a single unit, tRNA(Ile) is produced as an individual transcript and the 23S and 5S rDNAs are co-transcribed. Several of the features of this rDNA organization are unique, not having been described previously in any other eubacteria.

Molecular cloning and sequencing of infC, the gene encoding translation initiation factor IF3, from four enterobacterial species.

Translation initiation factor IF3 plays a crucial role in initiation of protein synthesis in bacteria. In order to elucidate the IF3 structural elements required for these functions, the evolutionary conservation of IF3 and its gene, infC, was investigated. Homologous infC sequences from Salmonella typhimurium, Klebsiella pneumoniae, Serratia marcescens and Proteus vulgaris were amplified by the polymerase chain reaction and sequenced. Analysis of these sequences, as well as that from Bacillus stearothermophilus, revealed several regions (e.g. residues 62-73 and 173-177) of absolute sequence conservation, suggesting an important role for these regions in IF3 function.

Structure-function analysis of Escherichia coli translation initiation factor IF3: tyrosine 107 and lysine 110 are required for ribosome binding.

Translation initiation factor IF3 is required for peptide chain initiation in Escherichia coli. IF3 binds directly to 30S ribosomal subunits ensuring a constant supply of free 30S subunits for initiation complex formation, participates in the kinetic selection of the correct initiator region of mRNA, and destabilizes initiation complexes containing noninitiator tRNAs. The roles that tyrosine 107 and lysine 110 play in IF3 function were examined by site-directed mutagenesis. Tyrosine 107 was changed to either phenylalanine (Y107F) or leucine (Y107L), and lysine 110 was converted to either arginine (K110R) or leucine (K110L). These single amino acid changes resulted in a reduced affinity of IF3 for 30S subunits. Association equilibrium constants (M-1) for 30S subunit binding were as follows: wild-type, 7.8 x 10(7); Y107F, 4.1 x 10(7); Y107L, 1 x 10(7); K110R, 5.1 x 10(6); K110L, < 1 x 10(2). The mutant IF3s were similarly impaired in their abilities to specifically select initiation complexes containing tRNA(fMet). Toeprint analysis indicated that 5-fold more Y107L or K110R protein was required for proper initiator tRNA selection. K110L protein was unable to mediate this selection even at concentrations up to 10-fold higher than wild type. The results indicate that tyrosine 107 and lysine 110 are critical components of the ribosome binding domain of IF3 and, furthermore, that dissociation of complexes containing noninitiator tRNAs requires prior binding of IF3 to the ribosomes.

Growth rate regulation of translation initiation factor IF3 biosynthesis in Escherichia coli.

infC, the gene encoding translation initiation factor IF3 in Escherichia coli, can be transcribed from three promoters. Two of these promoters, PI1 and PI2, are located in the upstream thrS sequence which codes for threonyl-tRNA synthetase. Previous studies had shown that PI2 was the major promoter for infC. In the present study, the extent of transcription from PI1 and/or PI2 at a variety of steady-state growth rates was analyzed by promoter fusion studies. PI2 was the more active promoter (two- to threefold stronger than PI1) at all growth rates tested. A fusion plasmid containing both PI1 and PI2 exhibited a transcription level approximately equal to the sum of those observed with the fusion plasmids containing the individual promoters. The transcriptional activities of PI1 and PI2 did not change as the growth rate was varied from 0.3 to 1.7 doublings per h. In contrast, a fusion plasmid carrying the rrnB P1 promoter displayed the expected growth rate response. The steady-state concentrations of infC mRNA in cells grown at different rates were measured and found not to vary. These results indicate that the previously reported growth rate regulation of IF3 biosynthesis neither is accomplished by transcriptional control nor is a result of differential mRNA stability. In view of these results, the steady-state levels of IF3 in cells grown at a number of different growth rates were determined by quantitative immunoblotting. IF3 levels were found to vary with growth rate in a manner essentially identical to that observed for ribosomes. A model accounting for these results and describing a mechanism for coordinate growth rate-regulated expression of ribosomes and IF3 is presented.